The Notch receptor family is a class of evolutionarily conserved transmembrane receptors that transmit signals affecting development in organisms as diverse as sea urchins and humans. Notch receptors and their ligands, Delta and Serrate (known as Jagged in mammals), are transmembrane proteins with large extracellular domains that contain epidermal growth factor (EGF)-like repeats. The number of Notch paralogues differs between species. For example, there are four Notch receptors in mammals (Notch1-Notch4), two in Caenorhabditis elegans (LIN-12 and GLP-1) and one in Drosophila melanogaster (Notch). Notch receptors are proteolytically processed during transport to the cell surface by a furin-like protease at a site S1 on the N-terminal side of the transmembrane domain, producing an extracellular Notch (ECN) subunit and a Notch transmembrane subunit (NTM). These two subunits remain non-covalently associated and constitute the mature heterodimeric cell-surface receptor. Notch receptors and the Notch signaling pathway are reviewed, e.g., in Aster et al., Annu. Rev. Pathol. Mech. Dis. 3:587-613, 2008, and Bolos et al., Endocrine Reviews 28:339-363, 2007.
Notch2 ECN subunits contain 36 N-terminal EGF-like repeats followed by three tandemly repeated Lin 12/Notch Repeat (LNR) modules that precede the S1 site. Each LNR module contains three disulfide bonds and a group of conserved acidic and polar residues predicted to coordinate a calcium ion. Within the EGF repeat region lie binding sites for the activating ligands.
The Notch2 NTM comprises an extracellular region (which harbors the S2 cleavage site), a transmembrane segment (which harbors the S3 cleavage site), and a large intracellular portion that includes a RAM23 domain, six ankyrin repeats, a transactivation domain and a carboxy-terminal PEST sequence. Stable association of the ECN and NTM subunits is dependent on a heterodimerization domain (HD) comprising the carboxy-terminal end of the ECN (termed HD-N) and the extracellular amino-terminal end of NTM (termed HD-C). Before ligand-induced activation, Notch is maintained in a resting conformation by a negative regulatory region (NRR), which comprises the three LNRs and the HD domain. The crystal structure of the Notch2 NRR is reported in Gordon et al., (2007) Nature Structural & Molecular Biology 14:295-300, 2007.
Binding of a Notch ligand to the ECN subunit initiates two successive proteolytic cleavages that occur through regulated intramembrane proteolysis. The first cleavage by a metalloprotease (ADAM17) at site S2 renders the Notch transmembrane subunit susceptible to a second cleavage at site S3 close to the inner leaflet of the plasma membrane. Site S3 cleavage, which is catalyzed by a multiprotein complex containing presenilin and nicastrin and promoting γ-secretase activity, liberates the intracellular portion of the Notch transmembrane subunit, allowing it to translocate to the nucleus and activate transcription of target genes. (For review of the proteolytic cleavage of Notch, see, e.g., Sisodia et al., Nat. Rev. Neurosci. 3:281-290, 2002.)
Five Notch ligands of the Jagged and Delta-like classes have been identified in humans (Jagged1 (also termed Serrate1), Jagged2 (also termed Serrate2), Delta-like1 (also termed DLL1), Delta-like3 (also termed DLL3), and Delta-like4 (also termed DLL4)). Each of the ligands is a single-pass transmembrane protein with a conserved N-terminal Delta, Serrate, LAG-2 (DSL) motif essential for binding Notch. A series of EGF-like modules C-terminal to the DSL motif precede the membrane-spanning segment. Unlike the Notch receptors, the ligands have short cytoplasmic tails of 70-215 amino acids at the C-terminus. In addition, other types of ligands have been reported (e.g., DNER, NB3, and F3/Contactin). (For review of Notch ligands and ligand-mediated Notch activation, see, e.g., D'Souza et al., Oncogene 27:5148-5167, 2008.)
The Notch pathway functions during diverse developmental and physiological processes including those affecting neurogenesis in flies and vertebrates. In general, Notch signaling is involved in lateral inhibition, lineage decisions, and the establishment of boundaries between groups of cells (see, e.g., Bray, Molecular Cell Biology 7:678-679, 2006). A variety of human diseases, including cancers and neurodegenerative disorders, have been shown to result from mutations in genes encoding Notch receptors or their ligands (see, e.g., Nam et al., Curr. Opin. Chem. Biol. 6:501-509, 2002). The connection between unrestrained Notch signaling and malignancy was first recognized when a recurrent t(7;9)(q34;q34.3) chromosomal translocation which creates a truncated, constitutively active variant of human Notch1 was identified in a subset of human acute lymphoblastic leukemias (T-ALL) (see, e.g., Aster et al., Annu. Rev. Pathol. Mech. Dis. 3:587-613, 2008). In mouse models, Notch1 signaling has been shown to be essential for T cell development and that Notch1-mediated signals promote T cell development at the expense of B cell development (see, e.g., Wilson et al., J. Exp. Med. 194:1003-1012, 2001).
Notch2 is also involved in certain cancers. Particularly, Notch2 is overexpressed in B-cell chronic lymphocytic leukemia (B-CLL), which in turn leads to overexpression of CD23, a hallmark of B-CLL cells. (See Hubmann et al., Blood 99:3742-3747, 2002.) Both Notch1 and Notch2 are highly expressed in multiple myeloma cells (cancerous plasma B cells), and stimulation with ligand strongly increases tumor cell growth. (See Jundt et al., Blood 103:3511-3515, 2004.) Notch2 and downstream effectors are overexpressed in melanoma (see Hoek et al., Cancer Res. 64:5270-5282, 2004; Seykora et al., Am J Dermatopathol 25:6-11, 2003), and the Notch2 locus is recurrently amplified in melanoma cell lines (Jonsson et al., Oncogene, 26:4738-4748, 2007). In addition, numerous studies have linked aberrant Notch2 signaling to breast cancer and other solid tumors (reviewed by Leong and Karsay, Blood 107:2223-2233, 2006). Notch2 is also required for marginal zone B cell development. (See Pillai et al., Annu. Rev. Immunol. 23:161-196, 2005.)
Given the involvement of Notch signaling in a wide variety of human diseases it is clear that there continues to be a need for agents that regulate Notch signaling and that have clinical attributes that are favorable for development as therapeutic agents. The invention described herein meets this need and provides other benefits.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.